一种可反映人精子DNA损伤严重程度的流式细胞术的建立及评价

Establishment and evaluation of a flow cytometry technique reflecting the severity of human sperm DNA damage

目的:建立一种可反映人精子DNA损伤严重程度的流式细胞检测技术,并对其性能进行评价。方法:DNA损伤精子(单链DNA)与荧光染料吖啶橙(AO)结合后发红色荧光,而DNA完整精子(双链DNA)与AO结合后发绿色荧光。通过对1165例人精液样本正常精子群的红色荧光和绿色荧光峰图的分析,确定红色荧光的95%的平均上限和绿色荧光的5%的平均下限为十字象限门的红色荧光和绿色荧光界限,建立精子DNA碎片指数(DFI)的流式细胞检测技术,并分析其重复性和线性范围,确定正常生育男性的参考值。并对122例不育门诊男性的精子DFI、轻度DNA损伤(DFIm)和重度DNA损伤(DFIs)与精子浓度及活力的相关性进行分析。结果:利用十字象限门建立的流式细胞术,可以很明显地区分不同人群的精子DFI、DFIm和DFIs。高、中、低DFI的精液样本使用流式细胞术重复检测10次,所得CV均低于5%。精子DFI在8.93%~53.90%范围内有极好的相关性,相关系数r>0.99。基于274例正常生育男性的精子DFI结果,以95%上限确定正常参考值范围,正常生育男性精子DFI≤25.50%。精子DFI与精子浓度没有显著相关性,但与精子活动率和前向运动精子百分率呈显著负相关,且DFIs与精子活动率和前向运动精子百分率的相关性(r分别为-0.592和-0.543)明显高于DFIm(r分别为-0.323和-0.236)。精子活动率和前向运动精子百分率降低组的精子DFIs和DFIm均显著高于正常组,且两组DFIs的差异(P<0.01)显著高于DFIm(P<0.05)。结论:本研究建立的可反映精子DNA损伤严重程度的流式细胞术,相比于现有的存在一定缺陷的流式细胞术,更适合在临床常规工作中推广使用。重度精子DNA损伤指标DFIs可能与生殖的关系更为密切。

Objective:To establish a flow cytometry(FC)technique reflecting the severity of human sperm DNA and evaluate its performance.Methods:We analyzed the red and green fluorescence peaks of normal sperm in 1165 human semen samples,defined the average lower limit of 5%of green fluorescence and the average upper limit of 95%of red fluorescence as the red and green fluorescence limits of the four-quadrant gate,and established an FC technique for detection of the sperm DNA fragmentation index(DFI),analysis of its repeatability and linear range and determination of the reference value of normal fertile men.We also analyzed the correlations of the sperm DFI,mild DNA damage marker(DFIm)and severe DNA damage marker(DFIs)with sperm concentration and motility in 122 men from the Infertility Clinic of Zhongda Hospital.Results:With the established FC technique based on the four-quadrant gate,the sperm DFI,DFIm and DFIs were clearly distinguished among different groups of males,and the coefficients of variation obtained in 10 repeated examinations of the semen samples with a high,medium or low DFI using the FC technique were all lower than 5%.The sperm DFI showed a very good correlation within the range of 8.93%-3.90%(r>0.99).With the upper limit of 95% as the range of normal reference value,the sperm DFI of 274 of the normal fertile males was≤25.50%.The sperm DFI was remarkably negatively correlated with sperm motility and the percentage of progressively motile sperm(PMS)but exhibited no significant correlation with sperm concentration.The DFIs showed significantly higher related coefficients with sperm motility and PMS(r=-0.592 and-0.543)than DFIm(r=-0.323 and-0.236).Both DFIs and DFIm were markedly higher in the patients with decreased sperm motility and PMS than in the normal fertile men,the former even more significantly(P<0.01)than the latter(P<0.05).Conclusion:Compared with the existing FC technique,ours can reflect the severity of sperm DNA damage and is more suitable for clinical application.DFIs may be more closely related to male fertility.